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Team:Aix-Marseille/Notebook
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| + | <!-- ======= WEEK 6 ======= --> | ||
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| + | <span class="note-tag" id="week6"></span> | ||
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| + | <h3 class="panel-title">Week 6 : 08/04/2014 ➡ 08/10/2014 <div class="pull-right"></div></h3> | ||
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| + | <ul class="nav nav-tabs" role="tablist"> | ||
| + | <li class="active"><a href="#0728" role="tab" data-toggle="tab" class="date-notebook">07/28/14</a></li> | ||
| + | <li><a href="#0729" role="tab" data-toggle="tab" class="date-notebook">07/29/14</a></li> | ||
| + | <li><a href="#0730" role="tab" data-toggle="tab" class="date-notebook">07/30/14</a></li> | ||
| + | <li><a href="#0731" role="tab" data-toggle="tab" class="date-notebook">07/31/14</a></li> | ||
| + | <li><a href="#0801" role="tab" data-toggle="tab" class="date-notebook">08/01/14</a></li> | ||
| + | <li><a href="#0803" role="tab" data-toggle="tab" class="date-notebook">08/03/14</a></li> | ||
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| + | <div class="tab-pane fade in active" id="0804"> | ||
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Revision as of 13:10, 22 September 2014
Notebook
Week 1 : 06/30/2014 ➡ 07/06/2014 Preparation of strains
- W3110 Escherichia coli strain and DH5alpha bacteria were put in culture on Petri dish.
- Both strains were incubated in Erlenmeyer flasks at 37 ° C.
- Preparation to electrocompetence : W3110 strain was washed in a 10% glycerol solution to remove extracellular salts that are lethal in this processing. W3110 competent bacteria were then frozen and stored.
- Preparation to chemocompetence : working at cold temperature, bacteria were washed and resuspended with CaCl2. Again, bacteria were washed and resuspended with CaCl2 and glycerol. Cells were frozen at -80°C.
- Electrocompetence : W3110 bacteria were transformed with pKOBEG plasmid by electroporation and put in culture at 30°C on petri dish.
- Colonies were observed in W3110 control cultures. Transformed bacteria (W3110pKOBEG) were put in culture at 30°C.
- Chemocompetence : A heat shock at 37°C was made with DH5α strain and the bacteria were then transformed with pBba_JO4450 plasmid and put in culture on petri dish.
- W3110pKOBEG bacteria were washed and arabinose was added to provide the expression of the recombinase encoded by pKOBEG plasmid. They were then incubated at 42 °C in order to destroy the thermosensitive pKOBEG plasmid.
- Preparation of electrocompetence : W3110 bacteria expressing the recombinase were prepared to electrocompetence and frozen at -80 ° C.
No colony was observed in the petri dish where DH5α transformed bacteria were previously put in culture. - Chemocompetence : bacteria were prepared using a formerly thawed aliquot. A heat shock at 42°C were made on competent DH5α containing pBba-J04450. They were finally put in culture at 37°C.
- DH5α control : Colonies were observed in the “negative control” culture. These colonies did not express RFP (no red fluorescent colour could be observed).
The LB we used in our experiments were contaminated, leading to the growth of undesirable colonies. The transformation of W3110 using pKOBEG plasmid was succesfull. The recombinase was expressed and the pKOBEG plasmid was destroyed using a thermosensitive origin of replication.
Week 2 : 07/07/2014 ➡ 07/13/2014
- Supercompetent DH5alpha preculture was made.
- Electroporation of W3110 strain with J04450 plasmid (20ng) and the telltale without plasmid was made.
- Preparation of supercompetent cells
The plasmid concentration contained in the iGEM kit was enough to transform bacteria by electroporation.
Week 3 : 07/14/2014 ➡ 07/20/2014
- DH5α supercompetent bacteria were transformed using 3 ng of J04450 plasmid or 60 pg of J04450 plasmid. Both cultures showed colonies meaning transformations were successful.
- DH5α supercompetent bacteria were transformed with respectively 1,5 ng of the following plasmids :
- K864600 plasmid
- B0032 plasmid
- B0033 plasmid
- E1010 plasmid
- B0034 plasmid
- B0010 plasmid
- B0030 plasmid
- E0040 plasmid
- pT18 RelA
- pBAD Mesh1
- DH5α bacteria containing respectively the following plasmids were put in liquid culture in order to perform miniprep the day after.
- K864600 plasmid
- B0032 plasmid
- B0033 plasmid
- E1010 plasmid
- B0034 plasmid
- B0010 plasmid
- B0030 plasmid
- E0040 plasmid
- DH5α supercompetent bacteria were transformed with respectively :
- pSB1C3 K608006 Cm resistant plasmid
- pSB1C3 K823017 Cm resistant plasmid
- pSB1C3 J04500 Cm resistant plasmid
- pWW2179 (SH3)4 Cm resistant plasmid
- pWW2021 CusR-L-LZa Kan resistant plasmid
- pWW2181 Amp resistant plasmid
- As a negative control, a culture of empty bacteria and Ampicillin was made.
- After the Miniprep was performed on bacteria containing pSB1AC3-J04450 plasmid, an electrophoresis was realised. It shows one high molecular weight band. The plasmid pSB1AC3-J04450 was successfully purified. It is stocked at -20°C and the strain is conserved in glycerol at -80°C.
Note :
Petri dishes containing respectively bacteria transformed with pT18 RelA plasmid and bacteria transformed with pBAD Mesh1 plasmid showed so many colonies that it was not possible to transfer isolated colonies into liquid culture. So, new cultures of both transformed bacteria were made again using a technique leading to isolated colonies.- After the Miniprep was performed on bacteria containing K864600 plasmid to E0040 plasmid, the following electrophoresis* was obtained :
* Be aware that Well 1 was filled with 5μL whereas the others were filled with only 3μL.
- Well 1: B0010
- Well 2: B0030
- Well 3: B0032
- Well 4: B0033
- Well 5: B0034
- Well 6: E0040
- Well 7: E1010
- Well 8: K864600
- Petri dish containing the bacteria transformed the day before with p5B1C3 K608006 Cm resistant plasmid to DH5α
- DH5α supercompetent bacteria previously transformed ( on 07/17/2014) with pSB1C3 K608006 Cm resistant plasmid to p5B1C3 K608006 Cm resistant plasmid were put in liquid culture in order to perform miniprep the day after
Week 4 : 07/21/2014 ➡ 07/27/2014
- Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
- pSB1C3 K608006
- pSB1C3 K823017
- pSB1C3 J04500
- pWW2179 (SH3)4 Cm resistant
- pWW2021 CusR-L-LZa Kan resistant
- pWW2181 Amp resistant
- pT18 RelA Amp resistant
- pBAD Mesh1 Amp resistant
Each well contains one or two fragments of DNA (corresponding to the different plasmid compaction degrees), thus all plasmids were successfully purified. They were stocked at -20°C and strains were put in glycerol at -80°C. - Bacteria transformed with the pSB1A2 BBa_B0010 plasmid were strangely red. That's why the plasmid was twice digested with EcoR1 and with XBa1 and Pst1. Digestion products were tested thanks to agarose gel electrophoresis. Results are presented below.
The digestion with EcoR1 made the plasmid linear. The digestion with Xba1 and Pst1 was supposed to produce two bands, corresponding to the backbone (2070pb) and to the BBa_B0010 part (80pb). No low molecular weight band appeared, the only visible band has a too low molecular weight to be the expected backbone. Something is wrong. We have to clarify this. Further experiments should be made to clarify these surprising results.
- The pSB1A2 BBa_B0010 plasmid was anew twice digested with EcoR1 and with XBa1 and Pst1. Digestion products are tested thanks to agarose gel electrophoresis. Results are presented below.
Again, these results did not make sense. It looks as if restriction enzymes did not cut the plasmid. This part is set-aside for the moment.
- pSB1C3 BBa_E1010, pSB1A2 BBa_E0040 and pSB1A2 BBa_B0034 plasmids were digested and ligated according to the BioBrick Assembly Kit of BioLabs. The destination plasmid DNA used is the backbone pSB1K3.m1 linearized plasmid. DH5α supercompetent bacteria were transformed with ligation products and spread over Kan dish
- DH5α supercompetent bacteria were transformed with BBA_J23100 (17D Plate 4) and spread over Amp dish.
- DH5α supercompetent bacteria previously transformed (on 23/17/2014) with BBA_J23100 Amp resistant plasmid and with Brick 200 and 202 Kan resistant plasmid were put in liquid culture in order to perform miniprep the day after
- Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
- Brick 200 (1)
- Brick 200 (2)
- Brick 202 (1)
- Brick 202 (2)
- J23100
Each well contains DNA fragments, thus all plasmids were successfully purified. - Bricks 200 (1), 200 (2), 202 (1) and 202 (2) were digested by EcoR1 and Spe1. Digestion products are tested thanks to agarose gel electrophoresis. Results are presented below.
To control it was the correct insert, a second electrophoresis was made with the RFP and GFP digestion products. Results are presented below.
Brick 200 (1), 200 (2) and 202 (1) did not present any insert. Only Brick 200 (2) seemed to present an insert.
Results were not conclusive. A control PCR might be made in order to determine the presence or the absence of BBa_B0034 RBS.
Week 5 : 07/28/2014 ➡ 08/03/2014
-
Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).
# DNA Template Primers Numbers Primers Names Weight of amplified DNA 2 Genomic DNA of W3110 6 - 7 serA_mut_ A849G_up
serA_tronq_SufRFC23_down171 bp 3 Genomic DNA of W3110 5 - 8 serA_PrefRFC10RBS_up
sera_mut_A849_down861 bp 6 Genomic DNA of W3110 10 - 13 cheA_SufRFC23_down
cheA_mut_T1290C_up726 bp 8 PiGEM_02.05 16 - 18 relA_mut_C1366_down
relA_sans_stop_SufRFC23_down882 bp 9 PiGEM_02.05 16 - 19 relA_mut_C1366_down
relA_2_stop_SufRFC23_down885 bp 10 PiGEM_02.06 20 - 24 Mesh_1_mut_A255T_up
Mesh_1_RFC10-RBS_up267 bp 11 PiGEM_02.06 21 - 22 Mesh_1_mut_A255T_down
Mesh_1_mut_A343G_up120 bp 12 PiGEM_02.06 23 - 25 Mesh_1_mut_A343G_down
Mesh1_sans_stop_SufRFC23_down192 bp 13 PiGEM_02.06 23 - 26 Mesh_1_mut_A343G_down
Mesh_1_2_stop_SufRFC23_down192 bp The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.
All DNA fragments seemed to be correctly amplified.
- PCR products were purified thanks to Macherey Nagel PCR Clean up Kit.
-
The DNA concentration of PCR products was tested thanks to the Nanodrop. Results are presented bellow.
# Concentration of DNA (ng/µL) 2 52.28 3 44.86 6 49.93 8 73.38 9 76.77 10 57.00 11 39.87 12 50.88 13 62.42 - PCR products number 6, 8 and 9 were stocked at -20°C whereas PCR products number 2, 3, 10, 11, 12 and 13 were used to realize SLIC protocol. This protocol permits to assemble all parts of SerA and Mesh1 genes with their directed mutation. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
-
Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
- Brick 200 (3)
- Brick 200 (4)
- Brick 200 (5)
- Brick 200 (6)
-
The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented following.
Brick 200 (6) well did not present any DNA fragment. Other wells contain one or two fragments of DNA, so Brick 200 (3), 200 (4) and 200 (5) were successfully purified. -
Several control PCR were realized to verify if SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.
# DNA Template Primers Numbers Primers Names Weight of amplified DNA S1 Clone 1 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
serA_tronq_SufRFC23_down1032 bp S2 Clone 2 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
serA_tronq_SufRFC23_down1032 bp S3 Clone 3 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
serA_tronq_SufRFC23_down1032 bp S4 Clone 4 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
relA_sans_stop_SufRFC23_down579 bp S5 Clone 5 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
relA_sans_stop_SufRFC23_down579 bp S6 Clone 6 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
relA_sans_stop_SufRFC23_down579 bp S7 Clone 7 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
Mesh_1_2_stop_SufRFC23_down582 bp S8 Clone 8 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
Mesh_1_2_stop_SufRFC23_down582 bp S9 Clone 9 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
Mesh_1_2_stop_SufRFC23_down582 bp -
Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).
# DNA Template Primers Numbers Primers Names Weight of amplified DNA 1 pKD4 plasmid 1 - 2 sdaCsdaB_mut_pKD4_up
sdaCsdaB_ mut_pKD4_down1032 bp 4 pKD4 plasmid 3 - 4 cheA_mut_pKD4_up
cheA_mut_pKD4_down1032 bp 5 Genomic DNA of W3110 9 - 14 cheA_PrefRFC23_up
cheA_mut_T1290C_down1032 bp 7 Genomic DNA of W3110 15 - 17 relA_mut_C1366G_up
relA_RFC10-RBS_up579 bp 
PCR product 1 and 4 were correctly amplified. Thus, they were purified thanks to Macherey Nagel PCR Clean up Kit. PCR product 5 and 7 seemed to be compound with too many different DNA fragments. That's why, a new amplification by PCR was made overnight (iGEM PCR protocol Q5 polymerase).
- Realization of "Day 2" of lambda red pKOBEG protocol. W3110 with active recombinase was electroporated by PCR product 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
-
The efficiency of the purification of PCR product number S1 to S9 was tested thanks to agarose gel electrophoresis. At the same time, the efficiency of the amplification of the beginning sequences of CheA and RelA (PCR product 5 and 7) was tested thanks to agarose gel electrophoresis. Results are presented bellow.
PCR products 5 and 7 were good for SLIC. Clone 1 and 2 (serA), clone 4 and 6 (Mesh1 without STOP) and clone 7 and 8 (Mesh1 with STOP) were put in culture in LB with Amp. -
The DNA concentration of PCR products 5 and 7 was tested thanks to the Nanodrop. Results are presented bellow.
# Concentration of DNA (ng/µL) 5 29.78 7 85.51 - PCR products number 5, 6, 7, 8 and 9 were used to realize SLIC protocol. This protocol permits to assemble all parts of CheA (5 and 6) and RelA (7 and 8 without STOP, 7 and 9 with STOP) genes with their directed mutation. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
- SLIC assembly of RelA was a success but SLIC assembly of CheA failed. That's why, a new SLIC assembly of CheA with PCR products 5 and 6 was made. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
- Realization of "Day 2" of lambda red pKOBEG protocol a second time. PCR product 1 and 4 were purified a second time. The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented bellow. W3110 with active recombinase was electroporated by PCR product 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
-
Several control PCR were realized to verify whether RelA SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.
# DNA Template Primers Numbers Primers Names Weight of amplified DNA S22 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
relA_sans_stop_SufRFC23_down2259 bp S23 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
relA_sans_stop_SufRFC23_down2259 bp S24 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
relA_sans_stop_SufRFC23_down2259 bp S25 Clone 25 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
relA_2_stop_SufRFC23_down2262 bp S26 Clone 26 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
relA_2_stop_SufRFC23_down2262 bp S27 Clone 27 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
relA_2_stop_SufRFC23_down2262 bp The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented bellow.
PCR products 1 and 4 were correctly amplified. Thus, they were purified thanks to Macherey Nagel PCR Clean up Kit. Transplanting of S22 to S27 failed, those clones were gave up. -
Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
- Clone 1 and 2 (SerA) ⇒ PiGEM_02.10 and PiGEM_02.11
- Clone 4 and 6 (Mesh1 without STOP) ⇒ PiGEM_02.12 and PiGEM_02.13
- Clone 7 and 8 (Mesh1 with STOP) ⇒ PiGEM_02.14 and PiGEM_02.15
- SLIC assembly of CheA failed. That's why, a new SLIC assembly of CheA with PCR products 5 and 6 was made. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
-
Several control PCR were realized to verify whether RelA SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.
# DNA Template Primers Numbers Primers Names Weight of amplified DNA S28 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
relA_sans_stop_SufRFC23_down2259 bp S29 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
relA_sans_stop_SufRFC23_down2259 bp S30 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
relA_sans_stop_SufRFC23_down2259 bp S31 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
relA_2_stop_SufRFC23_down2262 bp S32 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
relA_2_stop_SufRFC23_down2262 bp S33 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
relA_2_stop_SufRFC23_down2262 bp The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented bellow.
PCR product S28, S29, S31, S32 and S33 were good for SLIC. Clones 28 and 29 (RelA without STOP) and clones 31, 32 and 33 (RelA with STOP) were put in culture in LB with Amp. - Realization of "Day 2" of lambda red pKOBEG protocol for the third time. PCR products 1 and 4 were purified a third time. pKOBEG electrocompetent were prepared a third time. W3110 with active recombinase was electroporated with PCR products 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
-
The efficiency of the purification of PiGEM_02.10, PiGEM_02.11, PiGEM_02.12, PiGEM_02.13, PiGEM_02.14 and PiGEM_02.15 was tested thanks to agarose gel electrophoresis. Results are presented bellow.
These plasmids seemed to be in low quantity. The DNA concentration of plasmids was tested thanks to the Nanodrop. Results are presented following# Concentration of DNA (ng/µL) PiGEM_02.10 30.6 PiGEM_02.11 37.8 PiGEM_02.12 12.2 PiGEM_02.13 15.11 PiGEM_02.14 14.11 PiGEM_02.15 19.22
- The W3110 with active recombinase was electroporated with PCR products 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
- Bacteria transformed with PCR products 1 (sdaCsdaB) and 4 (cheA) put in culture on Kan dish on the 08/01/14 showed colonies.
- 5 different colonies of SdaCsdaB and CheA mutants were spread over Kan dish and Cm30 dish in order to verify whether the pKOBEG plasmid was eliminated (by the previously heat shock) or not as we wanted to, and whether the transformed bacteria contained the kanamycin resistance. The bacteria we want must show colonies on Kan dish and no colonies on Cm 30 dish at the same time.
Week 6 : 08/04/2014 ➡ 08/10/2014
